Reagents for the clonal isolation/enrichment of stem cell populations with mesenchymal tissue differentiation capacity are currently limiting. In this application, we propose a systematic effort to produce cell surface marker antibodies for these cells. These antibodies would provide the means to sort and purify distinct cell populations allowing meaningful gene expression studies to discover molecules important for the maintenance of stem/progenitor cells at different developmental stages. They will also allow the localization of stem cells in tissues. In addition, these markers may allow isolation of sufficient stem cells for ex vivo tissue engineering, or even within the operating room, such that a concentration of cells with differentiation capacity can be used for repair or regeneration of skeletal tissues without need for further in vitro isolation and expansion. We propose the use of a combination of techniques including a subtractive immunization protocol, a hybridoma growth method that allows management of large numbers of clones and screening methods that should allow discrete selection of relevant antibodies. We hypothesize that this approach will greatly increase the likelihood of generating specific adult stem cell antibodies over previously used methods. Our specific aim is to use subtractive immunizations to generate monoclonal antibodies against single cell suspensions of adult stem cells. Large scale screening will be done to identify antibodies that define important classes of stem cells. Sorted populations will be characterized for their properties of expandability and self-renewal. Novel antibodies to interesting stem cell populations will be identified. Specific antibody markers for stem/progenitor cells with the capacity to differentiate into mesenchymal tissues are lacking. The aim of this proposal is to develop novel antibodies that can be used to identify and sort these cells for the study of stem cell biology as well as for the purification of cells before therapeutic transplantation or before differentiation and subsequent transplantation. [unreadable] [unreadable] [unreadable]